Biocidal proteins

ABSTRACT

Biocidal proteins isolated from seeds have been characterised, in particular proteins isolated from members of the Brassicaceae, Compositae and Leguminosae families including Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus and Clitoria. The proteins show a wide range of antifungal activity and some are active against Gram-positive bacteria. All share a common amino acid sequence. DNA encoding the proteins has been isolated and incorporated into vectors. Plants transformed with this DNA may be produced. The proteins find commercial application as antifungal or antibacterial agents; transformed plants will show increased disease-resistance.

This is a division of application Ser. No. 08/377,687, filed Jan. 25,1995, now U.S. Pat. 5,538,525 which is a continuation of applicationSer. No. 08/002,480 filed Jan. 4, 1993, abandoned, which is acontinuation of PCT/GB92/01570 filed Aug. 27, 1992.

BACKGROUND OF THE INVENTION

This invention relates to biocidal proteins, processes for theirmanufacture and use, and DNA sequences coding for them. In particular,it relates to antimicrobial proteins isolated from seeds such as thoseof members of the Brassicaceae, Compositae or Leguminosae families.

In this context, antimicrobial proteins are defined as proteinspossessing at least one of the following activities: antifungal activity(which may include anti-yeast activity); antibacterial activity.Activity includes a range of antagonistic effects such as partialinhibition or death.

The Brassicaceae is a large family of herbs and shrubs which grow widelyin tropical, sub-tropical and temperate regions. The Family Brassicaceaeis also known as the "Cruciferae". Raphanus sativus (radish) belongs tothis family and is cultivated widely as a vegetable.

Dahlia belongs to the Compositae and has been extensively cultivated asan ornamental garden plant. A number of hybrids are commerciallyavailable, belonging to the Dahlia merckii or Dahlia variablis species.Cnicus benedictus, another Compositae, is a native plant of theMediterranean regions and was once used as a tonic and a cure for gout.

Lathyrus and Clitoria belong to the Leguminosae family. Lathyrus hasbeen extensively cultivated as an ornamental garden plant, the mostwidely known being the sweet pea plant, Lathyrus odoratus. The genusClitoria is less well known to European gardeners; Clitoria ternatea wasoriginally introduced from the East Indies in the 1800s.

Although plants normally grow on substrates that are extremely rich infungal organisms, infection remains a rare event. To keep out potentialinvaders, plants produce a wide array of antifungal compounds, either ina constitutive or an inducible manner. The best studied of these arephytoalexins which are secondary metabolites with a broad antimicrobialactivity spectrum that are specifically synthesised upon perception ofappropriate defence-related signal molecules. The production ofphytoalexins depends on the transcriptional activation of a series ofgenes encoding enzymes of the phytoalexin biosynthetic pathway. Duringthe last decade, however, it has become increasingly clear that someplant proteins can play a more direct role in the control ofphytopathogenic fungi. Several classes of proteins with antifungalproperties have now been identified, including chitinases,beta-1,3-glucanases, chitin-binding lectins, zeamatins, thionins andribosome-inactivating proteins.

These proteins have gained considerable attention as they couldpotentially be used as biocontrol agents. The chitinases andbeta-1,3-glucanases have weak activities by themselves, and are onlyinhibitory to plant pathogens when applied in combination (Mauch et al,1988, Plant Physiol, 88, 936-942). The chitin-binding lectins can alsobe classified as rather weak antifungal factors (Broekaert et al, 1989,Science, 245, 1100-1102; Van Parijs et al, 1991, Planta, 183, 258-264).Zeamatin is a more potent antifungal protein but its activity isstrongly reduced by the presence of ions at physiological concentrations(Roberts and Selitnermikoff, 1990, G Gen Microbiol, 136, 2150-2155).Finally, thionins and ribosome-inactivating proteins are potentiallyhazardous since they are known to be toxic for human cells (Carrasco etal, 1981, Eur J Biochem, 116, 185-189; Vernon et al, 1985, Arch BiochemBiophys, 238, 18-29; Stirpe and Barbieri, 1986, FEBS Lett, 195, 1-8).

SUMMARY OF THE INVENTION

We have now purified a new class of potent antimicrobial proteins withbroad spectrum activity against plant pathogenic fungi and with someantibacterial activity, moderate sensitivity to ions and apparent lowtoxicity for cultured human cells.

According to the present invention, we provide antimicrobial proteinscapable of being isolated from seeds and in particular from members ofthe Brassicaceae, the Compositae or the Leguminosae families includingRaphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus orClitoria.

In further aspects, this invention comprises a vector containing a DNAsequence coding for a protein according to the invention. The DNA may becloned or transformed into a biological system allowing expression ofthe encoded protein.

The invention also comprises plants transformed with recombinant DNAencoding an antimicrobial protein according to the invention.

The invention also comprises a process of combating fungi or bacteriawhereby they are exposed to the proteins according to the invention.

A new class of potent antimicrobial proteins has been isolated fromseeds of the Brassicaceae, the Compositae, and the Leguminosae. Similarproteins may be found in other plant families, genera and species. Theclass includes proteins which share a common amino acid sequence andwhich show activity against a range of plant pathogenic fungi.

The antimicrobial proteins isolated from seeds of Raphanus sativus(radish) include two protein factors, hereafter called Rs-AFP1 (Raphanussativus--Antifungal Protein 1) and Rs-AFP2 (Raphanus sativus--AntifungalProtein 2) respectively. Both are oligomeric proteins, composed ofidentical 5 kDa subunits. Both proteins are highly basic and have pIvalues above 10. Similar antifungal proteins have been isolated fromother Brassicaceae, including Brassica napus (Bn-AFPs), Brassica rapa(Br-AFPs), Sinapis alba (Sa-AFPs) and Arabidopsis thaliana (At-AFP1).

The antimicrobial proteins isolated from seeds of Dahlia and Cnicusinclude four protein factors, hereafter called Dm-AMP1 (Dahliamerckii--Antimicrobial Protein 1), Dm-AMP2 (Dahliamerckii--Antimicrobial Protein 2), Cb-AMP1 (Cnicusbenedictus--Antimicrobial Protein 1) and Cb-AMP2 (Cnicusbenedictus--Antimicrobial Protein 2) respectively. The Dm-AMP proteinsmay be isolated from seed of the Dahlia genus. The Cb-AMP proteins maybe isolated from seed of the Cnicus genus. All four proteins are closelyrelated and are composed of 5 kDa subunits arranged as oligomericstructures. All four proteins are highly basic.

The antimicrobial proteins isolated from seeds of Lathyrus and Clitoriainclude three protein factors, hereafter called Lc-AFP ( Lathyruscicera--Antifungal Protein), Ct-AMP1 (Clitoria ternatea--AntimicrobialProtein 1) and Ct-AMP2 (Clitoria ternatea--Antimicrobial Protein 2)respectively. Lc-AFP may be isolated from seed of the Lathyrus genus.The Ct-AMP proteins may be isolated from seed of the Clitoria genus. Allthree proteins are composed of 5 kDa subunits arranged as oligomericstructures and are highly basic.

N-terminal amino acid sequence determination has shown that the aboveproteins isolated from the Brassicaceae, Compositae and Leguminosae areclosely related and can be classified as a single protein family.Between the different plant families, the protein sequences areapproximately 50% identical. These sequences enable manufacture of theproteins by chemical synthesis using a standard peptide synthesiser.

The antimicrobial proteins are partially homologous to the predictedprotein products of the Fusarium--induced genes pI39 and pI230 in pea(Pisum sativum--a member of the Leguminosae family) as described byChiang and Hadwiger, 1991 (Mol Plant Microbe Interact, 4, 324-331). Thishomology is shared with the predicted protein product of the pSAS10 genefrom cowpea (Vigna unguiculata--another legume) as described byIshibashi et al (Plant Mol Biol, 1990, 15, 59-64). The antimicrobialproteins are also partially homologous with the predicted proteinproduct of gene pI322 in potato (Solanum tuberosum--a member of theSolanaceae family) as described by Stiekema et al, 1988 (Plant Mol Biol,11,255-269). Nothing is known about the biological properties of theproteins encoded by genes pI39, pI230, pSAS10 or pI322 as only the cDNAhas been studied. However, the pI39, pI230 and pI322 genes are switchedon after challenge to the plant by a disease or other stress. It hasbeen proposed that the pSAS10 gene encodes a protein involved ingermination. Due to their sequence similarity with the antimicrobialproteins of the invention, the proteins encoded by the pI39, pI230,pSAS10 or pI322 genes may be useful as fungicides or as antibiotics.

The antimicrobial protein sequences show a lower degree of partialhomology with the sequences of a group of small α-amylase inhibitorsfound in the following members of the Gramineae: sorghum (Bloch andRichardson, 1991, FEBS Lett, 279:101-104), wheat (Colitta et al, 1990,FEBS Lett, 270:191-194) and barley (Mendez et al, 1990 Eur J Biochem,194:533-539). Such proteins, including SIα2 from sorghum andγ-1-purothionin from wheat, are known to inhibit insect α-amylase andare toxic to insect larvae. It is not known if these α-amylaseinhibitors show any antifungal or other antimicrobial activity: no otherdata on their biological activity has been reported. Due to theirsequence similarity with the antimicrobial proteins of the invention,the α-amylase inhibitor proteins may be useful as fungicides or asantibiotics.

A third antifungal protein has been isolated from radish seeds,hereafter called Rs-nsLTP (Raphanus sativus non-specific lipid transferprotein). It is a dimeric protein, composed of two identical 9 kDasubunits. Amino acid sequence determination has identified the 43N-terminal residues of Rs-nsLTP, and has shown it to be homologous withnon-specific lipid transfer proteins isolated from other plants (Arondeland Kadet, 1990, Experientia, 46:579-585) but not with the otherantimicrobial proteins discussed above. The Rs-nsLTP sequence enablesmanufacture of the protein by chemical synthesis using a standardpeptide synthesiser.

Knowledge of their primary structure, enables the production of DNAconstructs encoding the antimicrobial proteins. The DNA sequence may bepredicted from the known amino acid sequence or the sequence may beisolated from plant-derived DNA libraries.

Oligonucleotide probes may be derived from the known amino acid sequenceand used to screen a cDNA library for cDNA clones encoding some or allof the protein. cDNA clones encoding the Rs-AFPs have been isolated inthis way and sequenced. These same oligonucleotide probes or cDNA clonesmay be used to isolate the actual AFP, AMP or Rs-nSLTP gene(s) byscreening genomic DNA libraries. Such genomic clones may include controlsequences operating in the plant genome. Thus it is also possible toisolate promoter sequences which may be used to drive expression of theantimicrobial (or other) proteins. These promoters may be particularlyresponsive to environmental conditions (such as the presence of a fungalpathogen).

DNA encoding the antimicrobial proteins (which may be a cDNA clone, agenomic DNA clone or DNA manufactured using a standard nucleic acidsynthesiser) can then be cloned into a biological system which allowsexpression of the proteins. Hence the proteins can be produced in asuitable micro-organism or cultured cell, extracted and isolated foruse. Suitable micro-organisms include Escherichia coli and Saccharomycescerevisiae. The genetic material can also be cloned into a virus orbacteriophage. Suitable cells include cultured insect cells and culturedmammalian cells. The DNA can also be transformed by known methods intoany plant species, so that the antimicrobial proteins are expressedwithin the plant.

Plant cells according to the invention may be transformed withconstructs of the invention according to a variety of known methods(Agrobacterium Ti plasmids, electroporation, microinjection,microprojectile gun, etc). The transformed cells may then in suitablecases be regenerated into whole plants in which the new nuclear materialis stably incorporated into the genome. Both transformed monocot anddicot plants may be obtained in this way, although the latter areusually more easy to regenerate.

Examples of genetically modified plants which may be produced includefield crops, cereals, fruit and vegetables such as: canola, sunflower,tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum,tomatoes, mangoes, peaches, apples, pears, strawberries, bananas,melons, potatoes, carrot, lettuce, cabbage, onion.

The AFP, AMP and Rs-nsLTP proteins show a wide range of antifungalactivity, including anti-yeast activity, and the AMPs are also activeagainst Gram positive bacteria. The proteins are useful as fungicides orantibiotics. Exposure of a plant pathogen to an antimicrobial proteinmay be achieved by application of the protein to plant parts usingstandard agricultural techniques (eg spraying). The proteins may also beused to combat fungal or bacterial disease by expression within plantbodies.

All the antimicrobial proteins show surprisingly high activity: theyinhibit the growth of a variety of plant pathogenic fungi atsubmicromolar doses. Antifungal activity of the AMPs is only partiallydependent on the ionic conditions. The antifungal effect of the AFPs isnot affected by K⁺ ions at physiological concentrations (50 mM). Theantifungal effect of Rs-AFP1, but not Rs-AFP2, is antagonised by Ca²⁺ atphysiological concentrations (1 mM). Rs-nsLTP also inhibits growth of avariety of plant pathogenic fungi, but is less potent and more saltsensitive that the AFPs.

The antimicrobial proteins can be isolated and purified from appropriateseeds, synthesised artificially from their known amino acid sequence, orproduced within a suitable micro-organism by expression of recombinantDNA. The proteins may also be expressed within a transgenic plant.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be further understood by reference to the drawings, inwhich:

FIG. 1 shows the cation exchange chromatogram for the Raphanusantifungal proteins and the associated graph of fungal growthinhibition.

FIG. 2A shows the HPLC profile of purified Rs-AFP1.

FIG. 2B shows the HPLC profile of purified Rs-AFP2 and Rs-nsLTP.

FIG. 3 shows the cation exchange chromatogram for the B napus antifungalproteins and the associated graph of fungal growth inhibition.

FIGS. 4A and 4B show the HPLC profile of purified B napus antifungalproteins.

FIG. 5 shows the cation exchange chromatogram for B rapa antifungalproteins and the associated graph of fungal growth inhibition.

FIGS 6A and 6B show the HPLC profile of purified B rapa antifungalproteins.

FIG. 7 shows the cation exchange chromatogram for S alba antifungalproteins and the associated graph of fungal growth inhibition.

FIGS. 8A and 8B show the HPLC profile of purified S alba antifungalproteins.

FIG. 9 shows the cation exchange chromatogram for A thaliana antifungalprotein and the associated graph of fungal growth inhibition.

FIG. 10 shows the HPLC profile of purified A thaliana antifungalproteins.

FIG. 11 shows the cation exchange chromatogram for the basic extract ofDahlia merckii and the corresponding graph of antifungal activity.

FIG. 12 shows the reverse-phase HPLC profile of purified Dm-AMP1 andDm-AMP2.

FIG. 13 shows the cation exchange chromatogram for the basic extract ofCnicus benedictus and the corresponding graph of antifungal activity.

FIG. 14 shows the reverse-phase HPLC profile of purified Cb-AMP1.

FIG. 15 shows the reverse-phase HPLC profile of purified Cb-AMP2.

FIG. 16 shows the cation exchange chromatogram for the basic extract ofLathyrus and the corresponding graph of antifungal activity.

FIG. 17 shows the reverse-phase HPLC profile of purified Lc-AFP.

FIG. 18 shows the cation exchange chromatogram for the basic extract ofClitoria and the corresponding graph of antifungal activity.

FIG. 19 shows the reverse-phase HPLC profile of purified Ct-AMP1.

FIG. 20 shows the reverse-phase HPLC profile of purified Ct-AMP2.

FIG. 21 shows the amino acid sequences of Rs-AFP1, RS-AFP2 and therelated Brassicaceae proteins.

FIG. 22 shows the amino acid sequences of the Dm-AMPs and the Cb-AMPs.

FIG. 23 shows the amino acid sequences of Lc-AFP and Ct-AMP1.

FIGS. 24A, 24B and 24C show the alignment of the amino acid sequences ofRs-AFP1, Dm-AMP1, the Cb-AMPs, Lc-AFP, Ct-AMP1, sorghum SIα2, wheat γ1purothionin, and the predicted products of the pea genes pI230 and pI39,of the cowpea gene pSAS10, and of the potato gene p322.

FIGS. 25A and 25B show predicted DNA sequences for the Dm-AMP and Cb-AMPgenes.

FIG. 25C shows predicted DNA sequences for the Lc-AFP and Ct-AMP1 genes.

FIG. 26 shows the amino acid sequence of Rs-nsLTP. FIGS. 27A and 27Bshow the alignment of the amino acid sequences of Rs-nsLTP and variousplant non-specific lipid transfer proteins.

FIG. 28 is a graph of Rs-AFP2 in vivo activity.

FIG. 29 shows the full length cDNA sequence of Rs-AFP1.

FIG. 30 shows the truncated cDNA sequence of Rs-AFP2.

FIGS. 31A and 31B show the full length DNA sequence of PCR assisted sitedirected mutagenesis of Rs-AFP2.

FIG. 32 shows the expression vector pFRG7.

FIG. 33 shows the expression vector pFRG8.

DESCRIPTION OF PREFERRED EMBODIMENTS

The following Examples illustrate the invention.

EXAMPLE 1

Antifungal and antibacterial activity assays.

Antifungal activity was measured by microspectrophotometry as previouslydescribed (Broekaert, 1990, FEMS Microbiol Lett, 69:55-60). Routinely,tests were performed with 20 μl of a (filter-sterilized) test solutionand 80 μl of a suspension of fungal spores (2×10⁴ spores/ml) in halfstrength potato dextrose broth (1/2 PDB). Some tests were performedusing a suspension of mycelium fragments in a synthetic growth medium.The synthetic growth medium consisted of K₂ HPO₄ (2.5 mM), MgSO₄ (50μM), CaCl₂ (50 μM), FeSO₄ (5 μM), CoCl₂ (0.1 μM), CuSO₄ (0.1 μM), Na₂MoO₄ (2 μM), H₃ BO₃ (0.5 μM), KI (0.1 μM), ZnSO₄ (0.5 μM), MnSO₄ (0.1μM), glucose (10 g/l), asparagine (1 g/l), methionine (20 mg/l),myo-inositol (2 mg/l), biotin (0.2 mg/l), thiamine-HCl (1 mg/l), andpyridoxine-HCl (0.2 mg/l). Control microcultures contained 20 μl ofsterile distilled water and 80 μl of the fungal suspension.

Unless otherwise stated the test organism was Fusarium culmorum (strainIMI 180420) and incubation was done at 25° C. for 48 hours. Percentgrowth inhibition is defined as 100 times the ratio of the correctedabsorbance of the control microculture minus the corrected absorbance ofthe test microculture over the corrected absorbance at 595 nm of thecontrol microculture. The corrected absorbance values equal theabsorbance at 595 nm of the culture measured after 48 hours minus theabsorbance at 595 nm measured after 30 min.

Antibacterial activity was measured microspectrophotometrically asfollows. A bacterial suspension was prepared by inoculating softnutrient agarose (tryptone, 10 g/l; Seaplaque agarose (FMC), 5 g/l).Aliquots (80 μl) of the bacterial suspension (10⁵ colony forming unitsper ml) were added to filter-sterilized samples (20 μl) in flat-bottom96-well microplates. The absorbance at 595 nm of the culture wasmeasured with the aid of a microplate reader after 30 minutes and 24hours of incubation at 28° C. Percent growth inhibition was calculatedas described above for the antifungal activity assay.

EXAMPLE 2

Extraction of the basic protein fraction from Raphanus sativus seeds.

Ammonium sulphate fractionation of proteins precipitating in theinterval of 30 to 70% relative saturation was followed by heat treatmentto remove heat-labile proteins, and by isolation of the basic proteinfraction (pI>9) by passage over a Q-Sepharose (Pharmacia) anion exchangecolumn equilibrated at pH 9. The detailed methods are described below.

One kg of R sativus seeds (obtained from Aveve, Belgium) was ground in acoffee mill and the resulting meal was extracted for 2 hours at 4° C.with 2 liters of an ice-cold extraction buffer containing 10 mM NaH₂PO₄, 15 mM Na₂ HPO₄, 100 mM KCl, 2 mM EDTA, 2 mM thiourea, and 1 mMPMSF. The homogenate was squeezed through cheesecloth and clarified bycentrifugation (30 min at 7,000×g). Solid ammonium sulphate was added tothe supernatant to obtain 30% relative saturation and the precipitateformed after standing overnight at room temperature was removed bycentrifugation (30 min at 7,000×g). The supernatant was adjusted to 70%relative ammonium sulphate saturation and the precipitate formedovernight at room temperature collected by centrifugation (30 min at7,000×g). After redissolving the pellet in 400 ml distilled water thesolution was heated at 80° C. for 15 min. The coagulated insolublematerial was removed by centrifugation (30 min at 7,000×g) and thesupernatant was dialyzed extensively against distilled water usingtubing (SpectralPot, Spectrum, USA) with a molecular weight cut off of1,000 Da. After dialysis the solution was adjusted to 50 mM Tris-HCl (pH9) by addition of the ten-fold concentrated buffer, and subsequentlypassed over a Q-Sepharose Fast Flow (Pharmacia, Uppsala, Sweden) column(12×5 cm) in equilibrium with 50 mM Tris-HCl (pH 9). The proteinfraction passed through the column was dialyzed extensively againstdistilled water and adjusted to 50 mM sodium N-morpholinoethanesulphonicacid (Na-MES), pH6, by addition of the ten-fold concentrated buffer.

This material represents the basic heat-stable protein fraction of Rsativus seeds. Its further chromatographic purification is described inExample 3.

EXAMPLE 3

Purification of antifungal proteins from R sativus seeds.

The starting material for the isolation of the R sativus antifungalproteins was the basic heat-stable protein fraction extracted from themature seeds as in Example 2. These proteins were further separated bycation exchange chromatography, as shown in FIG. 1.

About 150 mg of the basic heat-stable protein fraction dissolved in 50mM sodium MES (pH 6) was applied on a S-Sepharose High Performance(Pharmacia) column (10×1.6 cm) previously equilibrated with the sodiumMES buffer. The column was eluted at 2.5 ml\min with a linear gradientof 1000 ml from 0 to 500 mM NaCl in 50 mM sodium MES buffer (pH 6). Theeluate was monitored for protein by online measurement of the absorbanceat 280 nm (results shown in the lower panel of FIG. 1) and collected in10 ml fractions. Of these fractions, 20 μl was tested in themicrospectrophotometric antifungal activity assay described in Example 1using either the synthetic growth medium (Medium A: results shown asfull lines in the upper panel of FIG. 1) or the same medium supplementedwith 1 mM CaCl₂ and 50 mM KCl (Medium B: results shown as dashed linesin the upper panel of FIG. 1).

Upon fractionation, the mixture yielded a broad peak representing theunbound fraction, two well resolved peaks (peak 1 and peak 2) elutingaround 100 and 200 mM NaCl respectively, and a group of fivenon-resolved peaks (peaks 3 to 7) eluting between 250 and 450 mM NaCl.No antifungal activity was associated with the unbound fraction, whereasall bound peak fractions displayed antifungal activity when assayed inmedium A. However, tests performed in medium B only indicated growthinhibition for the fractions corresponding to peaks 1 and 2,respectively. It appears therefore that the antifungal activity of thesefractions is less salt-dependent than that of the fractions from peaks 3to 7.

The fractions showing antifungal activity in growth medium B (peaks 1and 2) were further purified by reversed-phase chromatography. About 1mg amounts of peak 1 material (FIG. 2A) and peak 2 material (FIG. 2B)were loaded on a Pep-S (porous silica C₂ /C₁₈, Pharmacia) column(25×0.93 cm) in equilibrium with 0.1% TFA. The column was eluted at 5ml/min with a linear gradient of 200 ml from 0.1% trifluoroacetic acid(TFA) to 40% acetonitrile/0.1% TFA. The eluate was monitored for proteinby online measurement of the absorption at 214 nm. Five ml fractions ofthe eluate were collected, vacuum-dried, and finally dissolved in 0.5 mldistilled water of which 10 μl was used in a microspectrophotometricantifungal activity assay.

FIG. 2A and FIG. 2B show the HPLC profiles of purified peak 1 and peak 2material respectively. The lower panels show monitoring of the eluatefor protein by measurement of the absorbance at 214 nm. Results of themicrospectrophotometric antifungal activity assay in medium A (fullline) and medium B (dashed line) are shown in the upper panels.

The material from peak 1 yielded a single major peak eluting at 30%acetonitrile and co-eluting with the antifungal activity in both mediumA and medium B. The active factor isolated from this peak is calledRs-AFP1 (Raphanus sativus antifungal protein 1). The peak 2 material, onthe other hand, resolved into two major peaks eluting at 30% and 33%acetonitrile respectively. The peak eluting at 30% acetonitrile wasactive in both medium A and medium B, whereas the peak eluting at 33%was active only in medium A. The active factor purified from the 30%acetonitrile peak is called Rs-AFP2 (Raphanus sativus antifungal protein2), and that from the 33% acetonitrile peak is designated Rs-nsLTP(Raphanus sativus non-specific lipid transfer protein) because of itshomology with non-specific lipid transfer proteins isolated from otherplant species (see Example 13).

EXAMPLE 4

Purity of the isolated Rs-AFPs.

The purity of the isolated antifungal proteins was verified by nativecathodic gel electrophoresis followed by protein staining and in situdetection of antifungal activity using a bio-zymographic technique.

Native cathodic gel electrophoresis and bio-zymography were done aspreviously described (De Bolle et al, 1991, Electrophoresis, 12,442-444) with some modifications. Electrophoresis was performed oncontinuous 10% acrylamide gels containing 60 mM Tris/70 mM MES (pH 7).The electrophoresis buffer consisted of 100 mM L-histidine/41 mM MES (pH6.5) Gels were cooled at 10° C. during electrophoresis. The samplescontained 20% glycerol, 0.0025% methylene blue, and 10 μg of purifiedRs-AFP1 or 20 μg of Rs-AFP2. Proteins were detected by silver-stainingof a diffusion blot prepared from the gel (Kovarik et al, 1987, FoliaBiological, 33, 253-257). The gel was overlaid with a soft agar gel (DeBolle et al, 1991, Electrophoresis, 12, 442-444) containing viableTrichoderma hamatum spores and incubated at 25° C. for 3 days.

Rs-ADP1and Rs-AFP2 migrate as single protein bands after cathodic gelelectrophoresis. Moreover, the antifungal activity co-migrated exactlywith the protein bands in the gel. These results indicate that theisolated factors are highly pure and that the antifungal activity is notattributable to minor contaminants.

EXAMPLE 5

Antifungal proteins related to Rs-AFPs from other species ofBrassicaceae.

Using the purification procedure described in Example 3, we haveisolated antifungal proteins from other Brassicaceae, including Brassicanapus, Brassica rapa, Sinapis alba and Arabidopsis thaliana.

FIG. 3 shows the cation exchange chromatogram for antifungal proteinisolated from B napus, and the associated graph of fungal growthinhibition (upper panel). FIGS. 4A and 4B show the HPLC profile of thepurified B. napus antifungal proteins, isolated from peak 1 (Bn-AFP1,FIG. 4A) and peak 2 (Bn-AFP2, FIG. 4B).

FIG. 5 shows the cation exchange chromatogram for antifungal proteinisolated from B rapa, and the associated graph of fungal growthinhibition (upper panel). FIGS. 6A and 6B show the HPLC profile of thepurified B rapa antifungal proteins, isolated from peak 1 (Br-AFP1, FIG.6A) and peak 2 (Br-AFP2, FIG. 6B).

FIG. 7 shows the cation exchange chromatogram for antifungal proteinisolated from S alba, and the associated graph of fungal growthinhibition (upper panel). FIGS. 8A and 8B show the HPLC profile of thepurified S alba antifungal proteins, isolated from peak 1 (Sa-AFP1, FIG.8A) and peak 2 (Sa-AFP2, FIG. 8B).

FIG. 9 shows the cation exchange chromatogram for antifungal proteinisolated from A thaliana, and the associated graph of fungal growthinhibition (upper panel). FIG. 10 shows the HPLC profile of the purifiedA thaliana antifungal proteins, isolated from peak 1 (At-AFP1).

All these antifungal proteins behave similarly to Rs-AFP1 and Rs-AFP2with respect to their SDS-PAGE and isoelectric focusing pattern (asdescribed in Example 5).

EXAMPLE 6

Extraction of the basic protein fraction from Dahlia merckii, Cnicusbenedictus, Lathyrus cicera and Clitoria ternatea seeds.

Five hundred grams of D merckii or C benedictus or Clitoria ternateaseeds (purchased from Chiltern Seeds, Cumbria, UK) or Lathyrus ciceraseeds (from Instituto Botanico Universitade Coimbra, Portugal) wereground in a coffee mill and the resulting meal was extracted for 2 hoursat 4° C. with 2 liters of an ice-cold extraction buffer containing 10 mMNaH₂ PO₄, 15 mM Na₂ HPO₄, 100 mM KCl, 2 mM EDTA and 1 mM benzamidine.The resulting homogenate was squeezed through cheesecloth and clarifiedby centrifugation (30 min at 7,000×g). Solid ammonium sulphate was addedto the supernatant to obtain 75% relative saturation and the precipitateallowed to form by standing overnight at 4° C. Following centrifugationat 7,000×g for 30 minutes, the precipitate was redissolved in a minimalvolume of distilled water and dialyzed extensively against distilledwater using benzoylated cellulose tubing (Sigma, St. Louis, Mo.). Afterdialysis the solution was adjusted to 50 mM NH₄ Ac (pH 9) by addition ofthe ten-fold concentrated buffer and passed over a Q-Sepharose Fast Flow(Pharmacia, Uppsala, Sweden) column (12×5 cm) equilibrated in 50 mM NH₄Ac (pH 9). The protein fraction which passed through the column wasadjusted to pH6 with acetic acid.

This material represents the basic (pI>9) protein fraction of the seeds.The fractions were further purified as described in Examples 7, 8, 9 and10.

EXAMPLE 7

Purification of antimicrobial proteins from Dahlia merckii seeds.

The starting material for the isolation of the D merckii antimicrobialproteins was the basic protein fraction extracted from the mature seedsas in Example 6. Proteins were further purified by cation exchangechromatography of this extract.

Approximately 500 ml of the basic protein fraction was applied to aS-Sepharose High Performance (Pharmacia) column (10×1.6 cm) equilibratedin 50 mM NH₄ Ac, pH 6.0. The column was eluted at 3.0 ml\min with alinear gradient of 50-750 ml NH₄ Ac, pH 6.0 over 325 minutes. The eluatewas monitored for protein by online measurement of the absorbance at 280nm (results shown in the lower panel of FIG. 11) and collected in 10 mlfractions. Samples from each fraction were assayed for antifungalactivity as described in Example 1 (results shown in the upper panel ofFIG. 11).

Following chromatography, the extract yielded a broad peak of activityeluting at around 250 mM NH₄ Ac. The fractions showing antifungalactivity were pooled and further purified by reverse-phase HPLC. About 3mg amounts of the peak were loaded on a PEP-S (porous silica C₂ /C₁₈,Pharmacia) column (25×0.4 cm) equilibrated with 0.1% TFA (trifluoraceticacid). The column was developed at 1 ml/min with a linear gradient of0.1% TFA to 100% acetonitrile/0.1% TFA over 100 minutes. The eluate wasmonitored for protein by online measurement of the absorption at 280 nm(results shown in the lower panel of FIG. 12). One ml fractions werecollected, vacuum-dried, and dissolved in 0.5 ml distilled water. 10 μlfrom each fraction was assayed for antifungal activity (results shown inthe upper panel of FIG. 12). The material yielded two well-resolvedpeaks of activity, eluting at 18% and 22% acetonitrile. These representthe purified proteins Dm-AMP1 and Dm-AMP2 respectively.

EXAMPLE 8

Purification of antimicrobial proteins from Cnicus benedictus seeds.

The procedure described in Example 7 was followed using the basicextract from Cnicus benedictus seeds. Following chromatography on theS-Sepharose High Performance column, the Cnicus extract yielded twopeaks of antifungal activity eluting at approximately 250 mM (peak 1)and 500 mM (peak 2) NH₄ Ac (results shown in FIG. 13).

Active fractions were pooled for each peak and further purified onreverse-phase HPLC as described in Example 7. Results for peak 1 areshown in FIG. 14: it yielded an active factor eluting at 18%acetonitrile which is designated Cb- AMP1. Similarly peak 2 eluted to asingle peak of activity which is designated Cb-AMP2 (results shown inFIG. 15).

EXAMPLE 9

Purification of antifungal protein from Lathyrus cicera seeds.

The procedure described in Example 7 was followed using the basicextract from Lathyrus cicera seeds. Following chromatography on theS-Sepharose High Performance column, the Lathyrus extract yielded asingle peak of antifungal activity eluting at approximately 160 mM NH₄Ac (results shown in FIG. 16).

Active fractions were pooled and further purified on reverse-phase HPLCas described in Example 7. Results for peak 1 are shown in FIG. 18: ityielded an active factor eluting at 22% acetonitrile which is designatedLc-AFP.

EXAMPLE 10

Purification of antimicrobial proteins from Clitoria ternatea seeds.

The procedure described in Example 7 was followed using the basicextract from Clitoria ternatea seeds. Following chromatography on theS-Sepharose High Performance column, the Clitoria extract yielded twopartially resolved peaks of antifungal activity eluting between 260 mMand 400 mM NH₄ Ac (results shown in FIG. 18).

Active fractions were pooled for each peak and further purified onreverse-phase HPLC as described in Example 7. Results for peak 1 areshown in FIG. 19: it yielded an active factor eluting at approximately18% acetonitrile which is designated Ct-AMP1. Similarly peak 2 yieldedan active factor eluting at approximately 18% acetonitrile which isdesignated Ct-AMP2 (results shown in FIG. 20).

EXAMPLE 11

Molecular structure of the purified antimicrobial proteins.

The molecular structure of the purified antimicrobial proteins wasfurther analysed. Sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE) was performed on precast commercial gels(PhastGel High Density from Pharmacia) using a PhastSystem (Pharmacia)electrophoresis apparatus. The sample buffer contained 200 mM Tris-HCl(pH 8.3), 1% (w/v) SDS, 1 mM EDTA, 0,005% bromophenol blue and, unlessotherwise stated, 1% (w/v) dithioerythritol (DTE). Proteins were fixedafter electrophoresis in 12.5% glutaraldehyde and silver-stainedaccording to Heukeshoven and Dernick (1985, Electrophoresis, 6,103-112).

The Rs-AFPs were analysed by SDS-PAGE. After reduction withβ-mercaptoethanol and modification of the cysteine residues byS-pyridylethylation, both Rs-AFP1 and Rs-AFP2 show single bands with anapparent molecular mass of about 5 kDa. After simple reduction withoutfurther cysteine derivatisation, the 5 kDa band is always accompanied bya 16 kDa band at variable yields, which may represent an oligomeric formof the 5 kDa protein resisted during electrophoresis. Unreduced Rs-AFP1and Rs-AFP2 migrate as single bands of 20 kDa and 17 kDa, respectively.These results show that the native Rs-AFPs are oligomeric proteins,consisting of dimers, trimers or tetramers of the 5 kDa polypeptide. Theoligomeric structure appears to be stabilised by disulphide linkages.SDS-PAGE analysis of unreduced Rs-AFP1, Rs-AFP1 reduced andS-pryridylethylated Rs-AFP2 with 200 ng of the protein separated on thegels. Myoglobin fragments were used as molecular weight markers(Pharmacia) with the following sizes: 17 kDa, 14.5 kDa, 8 kDa, 6 kDa,and 2.5 kDa.

SDS-PAGE analysis of Rs-nsLTP after reduction with DTE yielded a single9 kDa band. The unreduced Rs-nsLTP migrated as a single 18 kDa band. Itappears therefore that Rs-nsLTP is a dimeric protein (2×9 kDa)stabilised by disulphide bridges.

Purified Rs-nsLTP, reduced and non-reduced, was analyzed by SDS-PAGEwith molecular wright markers of myoglobin fragments described above.

Free cysteine thiol groups of the Rs-AFPs were assessed qualitatively asfollows. Hundred μg amounts of reduced or unreduced proteins weredissolved in 6M guanidinium-Cl containing 100 mM sodium phosphate buffer(pH 7) and 1 mM EDTA. The mixtures were allowed to react with5,5'-dithionitrobenzoic acid and monitored for release ofnitrothiobenzoate as described by Creighton (1989, Protein structure, apractical approach, 155-167). Reduction of the proteins was done byaddition of Tris-HCl (pH 8.6) to 100 mM and dithioerythritol to 30 mM,followed by incubation at 45° C. for 1 hour. The proteins were separatedfrom the excess reagents by reversed-phase chromatography on a C₂ /C₁₈silica column.

The unreduced Rs-AFPs did not contain free cysteine thiol groups,whereas the reduced proteins did, indicating that all cysteine residuesparticipate in disulphide bonds.

The pI values of Rs-AFP1 and Rs-AFP2 were determined by isoelectricfocusing and found to be higher than 10 for both proteins. Isoelectricfocusing was performed on precast Immobiline Dry Strips (Pharmacia)rehydrated in 8M urea, using marker proteins in the pI range from 4.7 to10.6 (Pharmacia).

When reduced with DTT, two purified proteins from Dahlia and twopurified proteins from Cnicus run as 5/6 kDa bands upon SDS-PAGEanalysis. In their unreduced form, the purified proteins run asoligomers. Unreduced Dm-AMP1 runs as a 24 kDa protein (FIG. 24, lane 1)and Dm-AMP2 as a 17 kDa protein. Similarly, unreduced Cb-AMP1 runs as asingle band of 30 kDa and Cb-AMP2 as a band of 18 kDa.

When reduced with DTT, SDS-PAGE analysis of three proteins two proteinspurified from Clitoria and one protein purified from Lathyrus run as 5/6kDa bands. In their unreduced form, the purified proteins run asoligomers. Unreduced Ct-AMP1 and Ct-AMP2 run as proteins ofapproximately 15 kDa whereas unreduced Lc-AFP runs as an approximately12 kDa protein.

EXAMPLE 12

Amino acid sequencing of the Rs-AFPs and related proteins.

Cysteine residues of the antifungal proteins were modified byS-pyridylethylation using the method of Fullmer (1984, Anal Biochem,142, 336-341). Reagents were removed by HPLC on a Pep-S (porous silicaC₂ /C₁₈) (Pharmacia) column (25×0.4 cm). The S-pyridylethylated proteinswere recovered by eluting the column with a linear gradient from 0.1%trifluoroacetic acid (TFA) to acetonitrile containing 0.1% TFA. Theresulting protein fractions were subjected to amino acid sequenceanalysis in a 477A Protein Sequencer (Applied Biosystems) with on-linedetection of phenylthiohydantoin amino acid derivatives in a 120AAnalyser (Applied Biosystems). Where necessary due to the proteins beingblocked, treatment of the S-pyridylethylated proteins with pyroglutamateamino peptidase was done according to the supplier's instructions(Boehringer Mannheim, Mannheim, FRG).

The N-terminal amino acid sequence of Rs-AFP1 and Rs-AFP2 was determinedby automated Edman degradation, after treatment with pyroglutamate aminopeptidase which cleaves off cyclic N-terminal glutamate residues. FIG.21 shows the sequence of the first 44 N-terminal amino acids of Rs-AFP1and of the first 35 residues of Rs-AFP2. The sequences of Rs-AFP1 andRs-AFP2 differ at only two positions within the first 36 residues. Thereplacement of a glutamic acid by a glutamine (position 4) and anasparagine by an arginine (position 27) in Rs-AFP2 are consistent withthe higher net positive charge of this protein relative to Rs-AFP1,which was previously evidenced by cathodic gel electrophoresis andcation exchange chromatography (FIG. 1). Rs-AFP1 appears to be rich incysteine and basic amino acids (5 and 9 respectively within the first 45residues). The molecular mass of Rs-AFP1 calculated on the basis of thepartial amino acid sequence (4964 Da) is very close to the valueestimated by SDS-PAGE (about 5000 Da) which indicates that thedetermined sequence encompasses the major part of the protein. However,it is anticipated that Rs-AFP1 contains at least one more cysteine,since the absence of free thiol groups assumes an even number ofcysteines.

FIG. 21 also shows the first 23 to 30 N-terminal amino acids of theRs-AFP-like proteins isolated from other Brassicaceae as described inExample 5 (Bn-AFP1, Bn-AFP2, Br-AFP1, Br-AFP2, Sa-AFP1, Sa-AFP2,At-AFP1SEQ ID NO:1through SEQ ID NO:9 ). All proteins were treated withpyroglutamate amino peptidase prior to sequencing but the cysteineresidues were not modified. Consequently, cysteine residues appear asblanks upon Edman degradation. Amino acids identical to thecorresponding amino acids in Rs-AFP1 are shown by dots. It appearstherefore that the Rs-AFP-like proteins from other members of the familyBrassicaceae are identical or nearly identical to Rs-AFP1 and Rs-AFP2.Br-AFP2 contains an unidentified uncommon amino acid at position 11.

FIG. 22 shows the complete amino acid sequence for the peptides Dm-AMP1,Cb-AMP1 and Cb-AMP2 (SEQ ID NO:10 through /seq ID NO:13. Shown also isthe sequence for the first 20 N-terminal amino acids of Dm-AMP2. Thesequences for Dm-AMP1 and Dm-AMP2 differ at only one position (position2) in these first 20 amino acids. Comparing the sequences for Cb-AMP1and Cb-AMP2, there are three changes. The substitution of an acidicresidue (aspartic acid at position 22) in Cb-AMP1 for a neutralasparagine in Cb-AMP2 and the substitution of glutamine at position 23for a basic lysine are consistent with the higher net positive charge.Similarly, Cb-AMP2 also differs from Dm-AMP1 at two positions althoughthe result is the net gain of two positive charges.

All four proteins show striking similarity to the proteins isolated fromseeds of the Brassicaceae family. Alignment of the amino acid sequencefor Rs-AFP1 (Raphanus Sativus--Antifungal Protein 1) with the sequencefor Dm-AMP1 reveals that they have approximately 50% identical residues.

FIG. 23 shows the complete amino acid sequence for the peptides Lc-AFPand Ct-AMP1(SEQ ID NO:14 and SEQ ID NO:15). Ct-AMP2 is expected to behighly homologous to Ct-AMP1. Both Lc-AFP and Ct-AMP1 are alsohomologous to the Compositae and Brassicaceae proteins. In particularCt-AMP1 is very homologous to the Dahlia peptide Dm-AMP1, having 35identical residues in its sequence.

Homologies can be found between this group of closely related proteinsand the products encoded by two pea (Pisum sativum) genes, pI39 andpI230, which are specifically induced by the fungus Fusarium solani(Chiang and Hadwiger, 1991, Mol Plant Microbe Interact, 4, 324-331), andwith the protein product of potato (Solanum tuberosum) gene p322(Stiekema et al, 1988, Plant Mol Biol, 11, 255-269). Nothing is knownabout the biological properties of the proteins encoded by genes pI39,pI230 or p322. In addition, theRs-AFP-like/Dahlia/Cnicus/lathyrus/Clitoria class of antimicrobialproteins show homology to inhibitors of insect gut α-amylases fromSorghum bicolor (Bloch and Richardson, 1991, FEBS Lett, 279, 101-104),and also to γ-purothionins from Triticum aestivum (Colilla et al, 1990,FEBS Lett, 270, 191-194) which inhibit in vitro protein synthesis incell-free systems (Mendez et al, 1990, Eur J Biochem, 194, 533-539).

FIGS. 24A, B and C show the alignment of the amino acid sequences ofRs-AFP1, Dm-AMP1, the Cb-AMPS, Lc-AFP, Ct-AMP1, the sorghum α-amylaseinhibitor SIα2, wheat γl purothionin, and the predicted sequences of themature protein products of the Fusarium-induced pea genes pI230 andpI39, of the cowpea gene pSAS10, and of the potato gene p322. Sequenceidentities and conserved changes compared with RS-AFP1 are boxed.Conserved changes are considered as substitutions within the amino acidhomology groups FWY MILV (SEQ ID NO:16), RHK, EDNQ (SEQ ID NO:17), andPAGST (SEQ ID NO:18). Gaps introduced for optimal alignment arerepresented by dashes (SEQ ID NO:19 through SEQ ID NO:30.

Upon alignment of the sequences, all of the cysteines and most of theglycines appear at conserved positions, suggesting their importance withrespect to structure and function of these proteins. Also noteworthy arethe conserved aromatic residues at positions 11 and 40.

FIGS. 25A and 25B show one of the possible DNA sequences of the genesencoding Dm-AMP1, Dm-AMP2, Cb-AMP1 and Cb-AMP2 (SEQ ID NO:31 through SEQID NO:34). Similarly FIG. 25C shows one of the possible DNA sequences ofthe genes encoding Lc-AFP and Ct-AMP1(SEQ ID NO:35 and SEQ ID NO:36).These gene sequences have been predicted from the known amino acidsequences using codons which commonly occur in dicotyledonous plants.The actual gene sequences within the seed may differ due to thedegeneracy of the genetic code.

EXAMPLE 13

Amino acid sequencing of Rs-nsLTP.

Amino acid sequencing of the Rs-nsLTP protein was carried out accordingto the description in Example 12.

FIG. 26 shows the first 43 N-terminal amino acids of Rs-nsLTP (SEQ IDNO:37) of which the cysteine residues were modified byS-pyridylethylation. In FIG. 27A and 27B the sequence of Rs-nsLTP isaligned with the N-terminal sequences of non-specific lipid transferproteins isolated from Spinacia oleracea (So-nsLTP; Bernhard et al,1990, Plant Physiol, 95, 164-170), Ricinus communis (Rc-nsLTP; Takishimaet al, 1986, Biochim Biophys Acta, 870, 248-255), Daucus carota(DC-nSLTP; Stenk et al, 1991, Plant Cell, 9, 907-921), Hordeum vulgate(Hv-nsLTP; Bernhard and Somerville, 1989, Arch Biochem Biophys, 269,695-697), and Zea mays (Zm-nsLTP; Tchang et al, 1988, J Biol Chem, 263,16849-16855). Gaps introduced for optimal alignment of the sequences areindicated by dashes (SEQ ID NO:38 through SEQ ID NO: 43). Identicalamino acids and conserved substitutions occurring in at least 4 of the 6sequences are boxed. Conserved changes are considered as substitutionswithin the amino acid homology groups FWY, MILV, RHK, EDNQ and PAGST.Rs-nsLTP shows 38 to 53% sequence identity with the non-specific lipidtransport proteins from other plant sources. Non-specific lipidtransport proteins are proteins that can translocate phospholipids orother apolar compounds between two membrane systems. These proteins werepreviously thought to play a role in the transport of phospholipids fromendoplasmic reticulum to cell and organelle membranes (Arondel andKaden, 1990, Experientia, 46, 579-585). However, recent evidence showsthat nsLTPs are located extra-cellularly, making their proposed functionin membrane biogenesis unlikely (Sterk et al, 1991, Plant Cell, 3,907-921).

EXAMPLE 14

Stability of the proteins' antifungal activity.

Tests for antifungal activity were performed with 20 μl samples dilutedfive-fold with growth medium containing Fusarium culmorum spores,according to the assay method given in Example 1. Untreated controlsamples consisted of the test proteins at 500 μg/ml in 10 mM sodiumphosphate buffer (pH 7). Heat stability tests were performed by heatingaliquots of the test proteins for 10 minutes at different temperaturesup to 100° C. Reduction of disulphide bridges was done by addition ofdithiothreitol at 30 mM and Tris-HCl (pH 8.6) at 300 mM. The reagentswere removed by reversed-phase chromatography. For digestions, differentproteases were added at 100 μg/ml and incubated at 37° C. for 16 hours.The control treatment containing only the reagents proved negative forantifungal activity after the reversed-phase chromatography step.

The antifungal activity of all the purified proteins tested wasresistant to heat treatments at up to 100° C. for 10 minutes. Reductionof their disulphide bonds by dithiothreitol, however, completelyabolished the antifungal activity. These disulphide linkages areessential for biological activity. Treatment of the Rs-AFP proteins withtrypsin, chymotrypsin, proteinase K or pronase E reduced the antifungalactivity by at least 10-fold.

EXAMPLE 15

Antifungal potency of the proteins.

The antifungal potency of the purified proteins was assessed ondifferent plant pathogenic fungi, using the assay described inExample 1. Growth of fungi, collection and harvest of fungal spores, andpreparation of mycelial fragments were done as previously described(Broekaert et al, 1990, FEMS Microbiol Lett, 69:55-60). The followingfungal strains were used: Alternaria brassicola MUCL 20297, Ascochytapisi MUCL 30164, Botrytis cinerea MUCL 30158, Cercospora beticola strainK897, Cladosporium sphaerosperum (K0791), Colletotrichum lindemuthianumMUCL 9577, Fusarium culmorum IMI 180420, Fusarium oxysporum f.sp. pisiIMI 236441, Fusarium oxysporum f.sp. lycopersici MUCL 909,Mycosphaerella fijiensis var fijiensis IMI 105378, Nectria haematococcaCollection Van Etten 160-2--2, Penicillium digitatum (K0879), Phomabetae MUCL 9916, Pyrenophora tritici-repentis MUCL 30217, Pyriculariaoryzae MUCL 30166, Rhizoctonia solani CBS 207-84, Sclerotiniasclerotianum MUCL 30163, Septoria nodorum MUCL 30111, Septoria tritici(K1097D), Trichoderma hamatum MUCL 29736, Trichoderma viride (K1127),Verticillium albo-atrum (K0937), Verticillium dahliae MUCL 19210,Venturia inaequalis MUCL 15927.

For C beticola, R solani, S sclerotianum, S nodorum and M fijiensis,mycelial fragments were used as inoculum, whereas all other fungi wereinoculated as spores.

Serial dilutions of the antifungal proteins were applied to the fungi,either using growth medium A or medium B. The percent growth inhibitionwas measured by microspectrophotometry. The concentration required for50% growth inhibition after 48 h of incubation (IC₅₀ value) wascalculated from the dose-reponse curves. The IC₅₀ values for the slowgrowing fungi S nodorum and V inaequalis was measured after 5 and 15days of incubation respectively.

The results for Rs-AFP1 and Rs-AFP2 are summarised in Table 1.

                  TABLE 1                                                         ______________________________________                                        ANTIFUNGAL ACTIVITY of Rs-AFP1 and Rs-AFP2                                             IC.sub.50 (μg/ml)                                                          Medium A     Medium B                                                Fungus     Rs-AFP1  Rs-AFP2   Rs-AFP1                                                                              Rs-AFP2                                  ______________________________________                                        A brassicola                                                                             15       2         >100   20                                       A pisi     5        4         >100   50                                       B cinerea  8        2         >100   >100                                     C beticola 2        2         100    3                                        C lindemuthianum                                                                         100      3         >100   >100                                     F culmorum 12       2         70     5                                        F oxysporum pisi                                                                         30       2         >100   >100                                     F oxysporum                                                                              15       2         >100   >100                                     lycopersici                                                                   M fijiensis                                                                              4        1.5       30     10                                       N haematococca                                                                           6        2         >100   30                                       P betae    2        1         20     6                                        P tritici-repentis                                                                       3        1.5       30     7                                        P oryzae   0.3      0.4       >100   7                                        R solani   100      >100      >100   >100                                     S sclerotianum                                                                           20       >100      >100   >100                                     S nodorum  20       15        100    20                                       T hamatum  2        2         20     4                                        V dahliae  5        1.5       >100   50                                       V inaequalis                                                                             ND       25        ND     >50                                      ______________________________________                                         ND = not determined                                                      

The concentration of Rs-AFPs required for 50% growth inhibition inmedium A varied from 0.3 μg/ml to over 100 μg/ml, depending on the testorganism. The antifungal potency of Rs-AFP1 is generally slightly lowerthan that of Rs-AFP2 in medium A. The difference in antifungal potencybetween Rs-AFP1 and Rs-AFP2 is more pronounced for the tests performedin medium B. Rs-AFP1 only inhibits 4 out of 17 fungi by more than 50% atconcentrations below 100 μg/ml, whereas Rs-AFP2 is inhibitory on 11 outof 18 fungi at this concentration. For some fungi, such as F culmorumand C beticola, the IC₅₀ value of Rs-AFP2 measured in medium A iscomparable to that obtained in medium B. On other fungi, such asFoxysporum f.sp. pisi, the IC₅₀ value of Rs-AFP2 is increased from 2μg/ml in medium A to over 100 μg/ml in medium B.

The antifungal potency of the Rs-AFP-like proteins from B napus, B rapa,S alba and A thaliana was compared to that of Rs-AFP1 and Rs-AFP2 usingfive different test fungi. The results of these experiments are shown inTable 2. With the exception of Br-AFP2, all proteins had specificactivities comparable to that of the Rs-AFPs. The fact that Br-AFP2 ison average 20-fold less active than the related species may be relatedto the observation that Br-AFP2 has an uncommon amino acid at position11 (see FIG. 21) whereas the Rs-AFPs and related proteins all have anaromatic residue at this position (see FIGS. 24A-C). When tested inmedium B, Rs-AFP2 appears to be the most potent protein, especially onthe fungus F culmorum.

                                      TABLE 2                                     __________________________________________________________________________    Antifungal Activity of Rs-AFP-like proteins from Brassica rapa,               Brassica napus, Sinapis alba and Arabidopsis thaliana                         Fungus                                                                              Rs-AFP1                                                                            Rs-AFP2                                                                            Br-AFP1                                                                            Br-AFP2                                                                            Bn-AFP1                                                                            Bn-AFP2                                                                            Sa-AFP1                                                                            Sa-AFP2                                                                            At-AFP1                         __________________________________________________________________________    IC.sub.50 (μg/ml) in medium A                                              A brassicola                                                                        15   2    3    75   0.60 1.20 1.2  4.5  10                              B cinerea                                                                           8    2    1.50 >100 2    2    1.8  3.5  3.90                            F culmorum                                                                          12   2    2    38   2.80 2.10 4    2.3  3                               F oxysporum                                                                         15   2    1.80 42   1.30 1.50 6    2.3  3                               lycopersici                                                                   P oryzae                                                                            0.3  0.4  0.25 3    0.35 0.25 0.5  0.3  0.25                            V dahliae                                                                           5    1.5  0.80 15   1.20 1    1.5  1.2  1.50                            IC.sub.50 (μg/ml) in medium B                                              A brassicola                                                                        >100 20   >100 >100 >100 >100 >100 >100 >100                            B cinerea                                                                           >100 >100 >100 >100 >100 >100 >100 >100 >100                            F culmorum                                                                          70   5    19   32   33   40   40   32   35                              F oxysporum                                                                         >100 >100 >100 >100 >100 >100 >100 >100 >100                            lycopersici                                                                   P oryzae                                                                            >100 7    >100 >100 32   8    25   3.8  >100                            V dahliae                                                                           >100 50   >100 >100 >100 >100 >100 >100 >100                            __________________________________________________________________________

The antifungal potency of Rs-nsLTP is shown in Table 3. On most fungiRs-nsLTP is 10 to 20 fold less potent relative to Rs-AFP2. Rs-nsLTP alsoappears to be highly salt-sensitive. None of the 13 fungi tested areinhibited by Rs-nsLTP in Medium B at concentrations below 100 μg/ml.

                  TABLE 3                                                         ______________________________________                                        Antifungal Activity of Rs-nsLTP                                                              IC.sub.50 (μg/ml)                                           Fungus           Medium A Medium B                                            ______________________________________                                        A brassicola     48       500                                                 A pisi           41       700                                                 B cinerea        45       680                                                 C lindemuthianum 25       >1000                                               F culmorum       20       520                                                 F oxysporum      54       >1000                                               lycopersici                                                                   F oxysporum pisi 58       900                                                 M fijiensis      >100     >100                                                N haematococca   100      >1000                                               F betae          18       750                                                 P oryzae         10       >1000                                               T hamatum        30       >1000                                               V dahliae        7        135                                                 ______________________________________                                    

The results for the Compositae proteins are summarised in Table 4.

The concentration of antimicrobial proteins required for 50% growthinhibition in medium A varied from 0.3 μg/ml to over 100 μg/ml,depending on the test organism. In general, the antifungal potency ofthe proteins was in the order: Cb-AMP2 >Cb-AMP1 >Dm-AMP1 >Dm-AMP2. Thedifferences in activity between the proteins is more pronounced inmedium B, with Cb-AMP2 showing the best salt tolerance. Dm-AMP1 andDm-AMP2 only inhibit the growth of 6 out of 11 fungi by more than 50% atconcentrations below 100 μg/ml, whereas the two Cnicus proteins inhibitthe growth of 7 out of 8 fungi when assayed in medium B.

Table 5 summarises the results for the antimicrobial proteins isolatedfrom Leguminosae seeds.

These proteins are active, although their activity is somewhat lowerthan that of the Rs-AFPs and Compositae proteins, especially whenassayed in high salt buffer, Medium B. In particular, the activity ofLc-AFP is markedly lower and comparable to the activity of Br-AFP2. Theamino acid sequence of Lc-AFP also shows a substitution at position 11which is normally tryptophan (FIGS. 21 and 23).

The high levels of antifungal activities demonstrated in vitro by eachof the purified proteins suggest that they may play a role in thedefence of seeds or seedlings against fungal attack.

                                      TABLE 4                                     __________________________________________________________________________    ANTIFUNGAL ACTIVITY of the Dm-AMPs and the Cb-AMPs                                     IC.sub.50 (μg/ml)                                                          Medium A              Medium B                                       FUNGUS   Dm-AMP1                                                                             Dm-AMP2                                                                             Cb-AMP1                                                                            Cb-AMP2                                                                            Dm-AMP1                                                                             Dm-AMP2                                                                             Cb-AMP1                            __________________________________________________________________________    A brassicola                                                                           1.1   2     ND   ND   140   140   ND                                 B cinerea                                                                              12    10    5    7    >200  >200  40                                 C beticola                                                                             1     3     1.2  1    6     6     5                                  C sphaerospermum                                                                       3     3     1    0.35 12    12    8                                  F culmorum                                                                             5     3     5    2    8     55    16                                 F oxysporum pisi                                                                       2.7   17    ND   ND   >200  >200  ND                                 P digitatum                                                                            2     2     2    1.4  70    50    15                                 P oryzae 5     6     ND   ND   >200  >200  ND                                 S tritici                                                                              1     0.5   0.8  0.5  4     2     2                                  T viride >80   >80   >100 40   >100  >100  >100                               V albo-atrum                                                                           4     2     ND   ND   ND    ND    ND                                 V dahliae                                                                              0.3   0.6   0.5  1.2  3     4     5                                  __________________________________________________________________________     ND = not determined                                                      

                                      TABLE 5                                     __________________________________________________________________________    ANTIFUNGAL ACTIVITY of the Ct-AMPs and LC-AFP                                          IC.sub.50 (μg/ml)                                                          Medium A      Medium B                                               FUNGUS   Ct-AMP1                                                                            Ct-AMP2                                                                            Lc-AFP                                                                            Ct-AMP1                                                                            Ct-AMP2                                                                            Lc-AFP                                       __________________________________________________________________________    B cinerea                                                                              37   15   80  >150 >150 >200                                         C sphaerospermum                                                                       9    3    10  >150 50   >200                                         F culmorum                                                                             18   6    20  75   50   >200                                         P digitatum                                                                            >150 >150 9   >150 >150 >200                                         P oryzae >150 >150 >200                                                                              >150 >150 >200                                         S tritici                                                                              9    2    37  >150 60   >200                                         T viride >150 >150 >200                                                                              >150 >150 >200                                         V albo-atrum                                                                           9    3    37  >150 100  >200                                         V dahliae                                                                              2    1    20  40   12   >200                                         __________________________________________________________________________

EXAMPLE 16

Effect of ions on antifungal activity.

The effect of ions on the antifungal activity of the Rs-AFPS andRs-nsLTP was examined in more detail. The IC₅₀ values of Rs-AFP1,Rs-AFP2 and Rs-nsLTP on F culmorum and T hamatum were measured in fivedifferent media. The reference medium was the synthetic growth mediumdescribed in Example 1 which contains a total of 2.5 mM monovalentcations and 0.1 mM divalent cations. The four other media contained 10mM KCl, 50 mM CaCl₂ or 5 mM CaCl₂ in supplement, respectively. For thepurpose of comparison, these tests were performed in parallel withβ-purothionin, an antifungal protein from wheat seeds (isolated asdescribed in Redman and Fisher, 1969, J Sci Food Agric, 20, 427-432) andMj-AMP2, an antifungal protein from Mirabilis jalapa seeds (Cammue etal, 1992, J Biol Chem, 267, 2228-2233).

Table 6 shows the results of the antifungal activity assays in thepresence of K⁺ and Ca²⁺.

Addition of KCl at up to 50 mM did not affect the antifungal activity ofeither Rs-AFP1 or Rs-AFP2. CaCl₂ at 1 mM had no effect on Rs-AFP2 butincreased the IC₅₀ value of Rs-AFP1 by about four-fold (ie, Ca²⁺ reducedthe antifungal activity of Rs-AFP1). CaCl₂ at 5mM almost completelyinactivated Rs-AFP1 while its effect on Rs-AFP2 varied from a slightincrease in IC₅₀ for F culmorum to complete inactivation for T hamatum.Addition of KCl at 50 mM decreases the activity of Rs-nsLTP by more than30-fold with both test fungi. In comparison, the IC₅₀ value ofβ-purothionin

                                      TABLE 6                                     __________________________________________________________________________    VARIATIONS IN ANTIFUNGAL ACTIVITY IN THE PRESENCE OF K.sup.+ AND              CA.sup.2+                                                                                  IC50 (μg/ml)                                                  Antifungal   Reference medium supplement:                                     Fungus                                                                              protein                                                                              None                                                                             10 mM K.sup.+                                                                       50 mM K.sup.+                                                                      1 mM Ca.sup.2+                                                                      5 mM Ca.sup.2+                               __________________________________________________________________________    F culmorum                                                                          Rs-AFP1                                                                              5  5     6    10    100                                                Rs-AFP2                                                                              3  2     2    2     5                                                  Rs-nsLTP                                                                             20 35    >1000                                                                              108   >1000                                              β-purothionin                                                                   10 7     4    10    70                                                 Mj-AMP2                                                                              4  5     40   50    >100                                         T hamatum                                                                           Rs-AFP1                                                                              7  7     7    30    >100                                               Rs-AFP2                                                                              2  2     3    2     >100                                               Rs-nsLTP                                                                             30 60    >1000                                                                              >1000 >1000                                              β-purothionin                                                                   4  3     1.5  4     30                                                 Mj-AMP2                                                                              2  2     25   20    >100                                         __________________________________________________________________________

increased by about 7-fold in the presence of 5 mM CaCl₂. Mj-AMP2appeared to be highly sensitive to the presence of salts, since its IC₅₀values increased by about 10-fold upon addition of either 1 mM CaCl₂ or50 mM KCl.

These results show that the Rs-AFPs are antagonised by divalent cations.Rs-AFP1 is much more sensitive to the presence of divalent cations thanRs-AFP2. Rs-nsLTP is clearly more salt-sensitive than either Rs-AFP1 orRS-AFP2. The antagonistic effect of cations appears to be stronglydependent on the test organism.

EXAMPLE 17

Effect of the purified antimicrobial proteins on the growth of theyeast, Saccharomyces cerevisiae.

The purified proteins were tested for their effect on Saccharomycescerevisiae. The method used was similar to the antifungal assaydescribed in Example 1 except that the growth medium was YPD (10 g/lyeast extract, 20 g/l bactopeptone, 20 g/l glucose) with 0.5% seaplaqueagarose.

When assayed at levels of 250 μg/ml, none of the purified Brassicaceaeproteins had an effect on the growth of Saccharomyces cerevisiae (strainSp1). Similarly, Lc-AFP did not inhibit the growth of S cerevisiae(strain JRY188) at a concentration of 200 μg/ml.

The Compositae and Clitoria peptides were active against the growth of Scerevisiae (strain JRY188). These results are shown in Table 7. Of thesix peptides, the two Clitoria peptides, Ct-AMP1 and Ct-AMP2 showed thehighest level of activity.

                  TABLE 7                                                         ______________________________________                                        ACTIVITY OF Dm-AMPs, Cb-AMPs and Ct-AMPs on YEAST                             Protein       IC.sub.50 (μg/ml)                                            ______________________________________                                        Dm-AMP1       50                                                              Dm-AMP2       50                                                              Cb-AMP1       30                                                              Cb-AMP2       20                                                              Ct-AMP1       18                                                              Ct-AMP2       9                                                               ______________________________________                                    

EXAMPLE 18

Effect of the purified antimicrobial proteins on bacteria.

The antibacterial effect of the purified proteins was assessed onAgrobacterium tumefaciens C58, Alcaligenes eutrophus, Azospirillumbrasilense Sp7, Bacillus megaterium ATCC 13632, Erwinia carotovorastrain 3912, Escherichia coli strain HB101, Pseudomonas solanacearumstrain K60 and Sarcina lutea ATCC 9342, using the assay described inExample 1.

Rs-AFP2 caused 50% inhibition in B megaterium at 200 μg/ml, but had noeffect on the other bacteria at concentrations up to 500 μg/ml.

The Compositae peptides Dm-AMP1, Dm-AMP2, Cb-AMP1 and Cb-AMP2 showedactivity only on B megaterium where they inhibited growth to 50% atconcentrations of 180, 40, 80 and 32 μg/ml respectively.

Rs-AFP1, Bn-AFPs, Br-AFP2, Sa-AFPs, Ct-AMPs and Lc-AFP had no effect onany of the bacteria at concentrations up to 500 μg/ml.

Results show that in general these proteins possess only weakantibacterial activity.

EXAMPLE 19

Effect of the purified antifungal proteins on cultured human cells.

Human cell toxicity assays were performed either on umbilical veinendothelial cells (Alessi et al, 1988, Eur J Biochem, 175, 531-540) orskin-muscle fibroblasts (Van Damme et al, 1987, Eur J Immunol, 17, 1-7)cultured in 96-well microplates. The growth medium was replaced by 80μlof serum-free medium (Optimem 1 for endothelial cells or Eagle's minimalessential medium (EMEM) for fibroblasts, both from GIBCO), to which 20μl of a filter-sterilised test solution was added. The cells werefurther incubated for 24 hours at 37° C. under a 5% CO₂ atmosphere with100% relative humidity. The viability of the cells was assessedmicroscopically after staining with trypane blue (400 mg/l in phosphatebuffered saline, PBS) for 10 minutes. Alternatively, cells were stainedwith neutral red (56 mg/l in PBS) for 2 hours at 37° C. Cells were lysedin acidic ethanol (100 mM sodium citrate, pH 4, containing 50% ethanol)and scored for release of the dye by microspectrophotometry at 540 nm.

The Rs-AFPs and Rs-nsLTP were evaluated for their potential toxiceffects using this assay. When added at up to 500 μg/ml to eithercultured human umbilical vein endothelial cells or human skin-musclefibroblasts, neither Rs-AFP1, Rs-AFP2, nor Rs-nsLTP affected cellviability after 24 h of incubation. In contrast, β-purothioninadministered at 50 μg/ml decreased the viability of both cell types bymore than 90%.

EXAMPLE 20

Anti-fungal activity of the Rs-AFPs against foliar disease: in vivo test

Rs-AFP2 was tested against the sugarbeet foliar disease Cercosporabeticola (strain E897) using the following method.

Sugarbeet plants were grown in John Innes potting compost (No. 1 or 2 )in 4 cm diameter mini-pots. The protein preparation was formulatedimmediately prior to use by dissolving in sterile distilled water anddiluting to the appropriate concentration. The formulation was appliedto the plants as a foliar spray. The spray was applied to maximumdiscrete droplet retention. Plants were treated with the protein one dayprior to inoculation with the disease which was applied as a foliarspray at a concentration of 50000 spores/ml. Plants were kept in anhumidity chamber for 48 hours and then transferred to the glasshouse.Disease was assessed following a further incubation of 8 days.

Results are shown in FIG. 28. The commercially available fungicidehexaconazole was used as a standard. Rs-AFP2 gave good control of thedisease and the concentration giving 50% control was approximately 15μM. In comparison hexaconazole gave 50% disease control when applied atapproximately 7 μM. This confirms that the protein can act as aneffective fungicide in vivo and that its activity is on a molar basiscomparable to the chemical standard.

EXAMPLE 21

Molecular cloning of RS-AFP1 and Rs-AFP2 cDNAs

From outdoor grown Raphanus sativus plants, seeds at 6 differentdevelopmental stages were collected, frozen in liquid nitrogen andstored at -80° C. After pulverisation, total RNA was extracted from 15 gof a mixture of the 6 different developmental stages, using the methodof De Vries et al (1988, Plant Molecular Biology Manual, B6, 1-13) withthe exception that 6 ml of a 1:2 phenol:RNA extraction buffer mixtureand 2 ml of chloroform were used per g of tissue. Poly (A)⁺ mRNA waspurified by affinity chromatography on oligo(dT)-cellulose as describedby Siflow et al (1979, Biochemistry 18, 2725-2731) yielding about 10 μgof poly(A)⁺ RNA per g of tissue. Double stranded cDNAs were preparedfrom 1,5 μg of poly(A)⁺ RNA according to Gubler and Hoffman (1983, Gene25, 263-269) and ligated to EcoRI/NotI adaptors using the cDNA SynthesisKit of Pharmacia. The cDNAs were cloned into the lambda ZAPII phagevector (Stratagene) according to the manufacturers instructions. A DNAprobe for screening the cDNA library was produced by polymerase chainreaction (PCR) as follows. Two degenerate oligonucleotides weresynthesised:

OWB15 (5'AAAGAATTCAARYTNTGYSARMGNCC 3'); SEQ ID NO:44 and

OWB17 (5'AAAGAATTCRTGNGCNGGRAANACRTARTTRC 3'); SEQ ID NO:45).

OWB15 corresponds to amino acids 2 to 7 of Rs-AFP1 and has a senseorientation. OWB17 corresponds to amino acids 36 to 43 of Rs-AFP1 andhas an antisense orientation. Both primers have the AAAGAATTC (i.e. AAAfollowed by the EcoRI recognition sequence) sequence at their 5' ends.PCR was performed with the Taq polymerase under standard conditions(Sambrook et al, 1989, Molecular Cloning, Cold Spring Harbor LaboratoryPress) using OWB15 and OWB17 as amplimers and 25 ng of cDNA as targetDNA. The temperature programme included an initial step at 94° C. for 5min, 30 cycles (94° C. for 1 min; 45° C. for 2 min, 72° C. for 3 min)and a final step at 72° C. for 10 min. The 144 bp PCR amplificationproduct was purified on a 3% agarose (NuSieve, FMC) gel. This PCRproduct was partially reamplified using the sense degenerateoligonucleotide OWB16 (5'AAAGAATTCGGNACNTGGWSNGGNGTNTG 3'; SEQ ID NO:46,and OWB17. OWB16 also has the AAAGAATTC (SEQ ID NO:47) extension at its5' end. This 123 bp PCR amplification product was again purified on a 3%agarose (NuSieve, FMC) gel and reamplified by PCR under the sameconditions except that the reaction mixture contained 130 μM dTTP and 70μM digoxigenin-11-dUTP instead of 200 μM dTTP. The digoxigenin-labeledPCR product was purified on a 3% NuSieve agarose gel. About 10,000plaque forming units of the lambda ZAPII cDNA library were screened withthe digoxigenin-labeled PCR product by in situ plaque hybridisationusing nylon membranes (Hybond-N, Amersham). Membranes were air-dried andDNA was crosslinked to the membranes under UV light (0.15 J/cm²).Hybridisation was performed for 16 h at 64° C. in 5×SSC, 1% blockingreagent (Boehringer Mannheim), 0.1% N-lauroylsarcosine, 0.02 % sodiumdodecylsulphate containing 10 ng/ml of heat denatureddigoxigenin-labeled probe. Non-specifically bound probe was removed byrinsing two times 5 min in 2×SSC/0.1% SDS at 25° C. and two times 15 minin 0.1×SSC/0.1% SDS at 60° C. Detection of the probe was done usinganti-digoxigenin antibodies linked to alkaline phosphatase (BoehringerMannheim) and its substrate 5-bromo-4-chloro-3-indolyl phosphate(Boehringer Mannheim) according to the manufacturers instructions.Positive plaques were purified by two additional screening rounds withthe same probe under the same conditions. Inserts from purified plaqueswere excised in vivo into the pBluescript phagemid form with the aid ofthe helper phage R408. The inserts from 22 different positive cloneswere excised by EcoRI digestion and their sizes compared by agarose gelelectrophoresis. Four clones had an insert of approximately 400 bp, theother 18 positive clones contained inserts ranging between approximately250 and 300 bp. The four clones with the 400 bp inserts and six cloneswith the smaller inserts were subjected to nucleotide sequence analysis.The clones with the largest insert all had an open reading frame of 80amino acids corresponding to Rs-AFP1, as could be determined bycomparison to the experimental N-terminal amino acid sequences (seeExample 12). The 243 bp open reading frames code for the mature Rs-AFP1(50 amino acids) preceded by a putative 29 amino acid signal sequenceobeying the (-1, -3) rule (von Heijne 1985, Mol. Biol. 184, 99-105).These full-length cDNA clones only differed from each other in thelength of their 5' and 3' end untranslated regions. Five of the cloneswith the smallest insert were partially identical to the full-lengthRs-AFP1 cDNA clones except that they were truncated at their 5' ends.The remaining clone was identified as a 5' truncated Rs-AFP2 cDNA cloneby comparing the deduced and the experimentally determined amino acidsequences. When comparing the full-length Rs-AFP1 cDNA clone pFRG1 (FIG.29; SEQ ID NO:48 and SEQ ID NO:49) and the truncated Rs-AFP2 cDNA clonepFRG2 (FIG. 30; SEQ ID NO:50 and SEQ ID NO:51), it can be seen that thecodon usage is slightly different and that the 3' end untranslatedregion of the Rs-AFP2 cDNA is longer than the one of the Rs-AFP1 cDNA.Finally, both the Rs-AFP1 and the Rs-AFP2 cDNA clones have at least twopolyadenylation signals. FIG. 29 shows the nucleotide sequence and thededuced amino acid sequence of the full-length Rs-AFP1 cDNA clone pFRG1.The putative signal sequence is underlined and the sequence of themature Rs-AFP1 is boxed. FIG. 30 shows the nucleotide sequence and thededuced amino acid sequence of the 5' truncated Rs-AFP2 cDNA clonepFRG2.

In order to obtain a full-length Rs-AFP2 CDNA, another approach wasfollowed: PCR was performed under standard conditions using theantisense oligonucleotide OWB23 (5'ATAGAATTCGACGTGAGCTTATCATCTTATTATCCG3'); SEQ ID NO:52, in combination with the M13 universal primer at onehand and the M13 reverse primer at the other hand. The last 30nucleotides of OWB23 form the inverted complementary sequence of thepart of the 3' untranslated region immediately flanking the poly-A tailof pFRG2 (see FIG. 30). This sequence is extended to the 5' end with theGAATTC EcoRI recognition site preceded by the nucleotides `ATA`. As atemplate, either 2 μg of total cDNA or 10⁵ recombinant phages were used.In both cases, 3 separate reactions were set up. Prior to amplification,phages were lysed by an initial step in the PCR temperature programme of5 min at 99° C. to liberate the phage DNA. The size of the amplificationproducts was determined by electrophoresis on a 3% agarose (NuSieve,FMC) gel. Products were obtained with sizes corresponding to inserts of280 to 300 bp. Thus, it can be concluded that no full-length Rs-AFP2cDNA clones seem to be present in the cDNA library.

EXAMPLE 22

Mutagenesis of RS-AFP1 CDNA to RS-AFP2 DNA

As can be deduced from the experimentally determined N-terminalsequences (see Example 12) and the nucleotide sequences (see Example21), Rs-AFP1 and Rs-AFP2 only differ in two amino acids as stated inExample 12. As the antifungal potency of Rs-AFP2 is significantly higherthan that of Rs AFP1 (see Table 1) and a full-length cDNA clone of theRs-AFP2 is not available, the Rs-AFP1 cDNA was transformed into theRs-AFP2 nucleotide sequence by PCR-assisted site-directed mutagenesisaccording to the method of E. Merino et al (1992, BioTechniques 12,508-510). The following oligonucleotides (SEQ ID NO:53 through SEQ IDNO:57) were used:

OWB28 (5'CTTGGCCTTTGGCACAACTTC 3'),

OWB29 (5'GCTTTCTCAAGTCTAATGCAC 3'),

OWB30 (5'AACTCGAGCTGCAGTGTCGACCTATTAACAAGGAAAGTAGC 3'),

OWB35 (5'GGAATAGCCGATCGAGATCTAGGAAACAGCTATGACCATG 3'),

OWB36 (5'GGAATAGCCGATCGAGATCTAGGA 3').

The first mutation (glutamate into glutamine at position 5 of the matureprotein) was introduced by performing PCR with the Pfu polymerase(Stratagene) using OWB35 (this is the M13 universal primer with a 5' tagsequence) and OWB28 (the first antisense mutagenesis primer) asamplimers and 100 ng of the KpnI-digested pFRG1 cDNA as target DNA.MgCl₂ was added to the amplification mixture to a final concentration of50 mM. The temperature programme included an initial step at 94° C. for5 min, 30 cycles (94° C. for 1 min, 45° C. for 2 min, 72° C. for 3 min)and a final step at 72° C. for 10 min. In a second step, this PCRproduct was used as a megaprimer and extended by the Pfu polymeraseusing 50 ng of the KpnI-digested pFRG1 cDNA as the target DNA. Thetemperature programme included an initial step at 94° C. for 5 minfollowed by a 5 cycles extension (94° C. for 1 min, 50° C. for 1 min,72° C. for 1 min). Then OWB29 (the antisense primer introducing thesecond mutation, from asparagine to arginine at position 27 of themature protein) and OWB36 (which is identical to the 5' tag sequence ofOWB35) were added, followed by PCR amplification by the Pfu-polymeraseas described for the introduction of the first mutation. To get afull-length Rs-AFP2 nucleotide sequence, the procedure outlined in thesecond step was repeated though using the oligonucleotide primers OWB36and OWB30 (which introduces a second stop codon followed by the SalI,PstI and XhoI restriction sites, thus also eliminating the 3' enduntranslated region of the Rs-AFP1 cDNA clone pFRG1). The final PCRproduct was cut with BamHI (occurring in the polylinker of thepBluescript phagemid pFRG1) and SalI, subcloned in pEMBL18+(pre-digested with the same restriction enzymes) and subjected tonucleotide sequence analysis. FIGS. 32A and B show the nucleotidesequence and the derived amino acid sequence of the full-length Rs-AFP2DNA clone pFRG4 obtained by PCR-assisted site-directed mutagenesis ofthe Rs-AFP1 cDNA clone pFRG1(SEQ ID NO:58 and SEQ ID NO:59). Theputative signal sequence is underlined and the sequence of the matureRs-AFP2 is boxed.

EXAMPLE 23

Construction of the expression vector pFRG7

The expression vector pFRG7 (FIG. 32; SP=signal peptide, MP=matureprotein) contains the full coding region of the Rs-AFP2 DNA flanked atits 5' end by the strong constitutive promoter of the 35S RNA of thecauliflower mosaic virus (Odell et al, 1985, Nature 313, 810-812) with aduplicated enhancer element to allow for high transcriptional activity(Kay et al, 1987, Science 236, 1299-1302). The coding region of theRs-AFP2 DNA is flanked at its 3' end side by the polyadenylationsequence of 35S RNA of the cauliflower mosaic virus (CaMV35S). Theplasmid backbone of this vector is the phagemid pUC120 (Vieira andMessing 1987, Methods Enzymol. 153, 3-11). pFRG7 was constructed asfollows: clone pFRG4 which consisted of the Rs-AFP2 DNA (FIG. 37) clonedinto the BamHI/SalI sites of pEMBL18+, Boehringer). The 298 bpBamHI/SalI fragment was subcloned into the expression vector pFAJ3002which was pre-digested with BamHI and SalI. pFAJ3002 is a derivative ofthe expression vector pFF19 (Timmermans et al, 1990, J. Biotechnol. 14,333-344) of which the unique EcoRI site is replaced by a HindIII site.

EXAMPLE 24

Construction of the plant transformation vector pFRG8

The expression vector pFRG7 was digested with HindIII and the fragmentcontaining the Rs-AFP2 DNA expression cassette was subcloned into theunique

HindIII site of pBin19Ri. pBin19Ri is a modified version of the planttransformation vector pBin19 (Bevan 1984, Nucleic Acids Research 12,8711-8721) wherein the unique EcoRI and HindIII sites are switched andthe defective nptII expression cassette (Yenofsky et al. 1990, Proc.Natl. Acad. Sci. USA 87: 3435-3439) is introduced. The new planttransformation vector is designated pFRG8 (FIG. 33).

EXAMPLE 25

Plant Transformation

The disarmed Agrobacterium tumefaciens strain LBA4404 (pAL4404)(Hoekemaet al, 1983, Nature 303, 779-180) was transformed with the vector pFRG8using the method of de Framond et al (BioTechnology 1, 262-269).

Tobacco transformation was carried out using leaf discs of Nicotianatabacum Samsun based on the method of Horsch et al (1985, Science 227,1229-1231) and co-culturing with Agrobacterium strains containing pFRG8.Co-cultivation was carried out under selection pressure of 100 μg/mlkanamycin. Transgenic plants (transformed with pFRG8) were regeneratedon media containing 100 μg/ml kanamycin. These transgenic plants may beanalysed for expression of the newly introduced genes using standardwestern blotting techniques. Plants capable of constitutive expressionof the introduced genes may be selected and self-pollinated to giveseed. F1 seedlings of the transgenic plants may be further analysed.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 59                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GlnLysLeuCysGluArgProSerGlyThrTrpSerGlyValCysGly                              151015                                                                        AsnAsnAsnAlaCysLysAsnGlnCysIleAsnLeuGluLysAlaArg                              202530                                                                        HisGlySerCysAsnTyrValPheProAlaHisLys                                          3540                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GlnLysLeuCysGlnArgProSerGlyThrTrpSerGlyValCysGly                              151015                                                                        AsnAsnAsnAlaCysLysAsnGlnCysIleArgLeuGluLysAlaArg                              202530                                                                        HisGlySerCys                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GlnLysLeuCysGluArgProSerGlyThrTrpSerGlyValCysGly                              151015                                                                        AsnAsnAsnAlaCysLysAsnGlnCysIleAsn                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GlnLysLeuCysGluArgProSerGlyThrXaaSerGlyValCysGly                              151015                                                                        AsnAsnAsnAlaCysLysAsnGlnCysIleArg                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GlnLysLeuCysGluArgProSerGlyThrTrpSerGlyValCysGly                              151015                                                                        AsnAsnAsnAlaCysLysAsnGlnCysIleAsnLeuGluLys                                    202530                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GlnLysLeuCysGluArgProSerGlyThrTrpSerGlyValCysGly                              151015                                                                        AsnAsnAsnAlaCysLysAsn                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GlnLysLeuCysGluArgProSerGlyThrTrpSerGlyValCysGly                              151015                                                                        AsnAsnAsnAlaCysLysAsnGlnCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GlnLysLeuCysGlnArgProSerGlyThrTrpSerGlyValCysGly                              151015                                                                        AsnAsnAsnAlaCysArgAsnGlnCysIle                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlnLysLeuCysGluArgProSerGlyThrTrpSerGlyValCysGly                              151015                                                                        AsnSerAsnAlaCysLysAsnGlnCysIleAsn                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GluLeuCysGluLysAlaSerLysThrTrpSerGlyAsnCysGlyAsn                              151015                                                                        ThrGlyHisCysAspAsnGlnCysLysSerTrpGluGlyAlaAlaHis                              202530                                                                        GlyAlaCysHisValArgAsnGlyLysHisMetCysPheCysTyrPhe                              354045                                                                        AsnCys                                                                        50                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GluValCysGluLysAlaSerLysThrTrpSerGlyAsnCysGlyAsn                              151015                                                                        ThrGlyHisCys                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GluLeuCysGluLysAlaSerLysThrTrpSerGlyAsnCysGlyAsn                              151015                                                                        ThrLysHisCysAspAspGlnCysLysSerTrpGluGlyAlaAlaHis                              202530                                                                        GlyAlaCysHisValArgAsnGlyLysHisMetCysPheCysTyrPhe                              354045                                                                        AsnCys                                                                        50                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GluLeuCysGluLysAlaSerLysThrTrpSerGlyAsnCysGlyAsn                              151015                                                                        ThrLysHisCysAspAsnLysCysLysSerTrpGluGlyAlaAlaHis                              202530                                                                        GlyAlaCysHisValArgSerGlyLysHisMetCysPheCysTyrPhe                              354045                                                                        AsnCys                                                                        50                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      LysThrCysGluAsnLeuSerGlyThrPheLysGlyProCysIlePro                              151015                                                                        AspGlyAsnCysAsnLysHisCysLysAsnAsnGluHisLeuLeuSer                              202530                                                                        GlyArgCysArgAspAspPheXaaCysTrpCysThrArgAsnCys                                 354045                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AsnLeuCysGluArgAlaSerLeuThrTrpThrGlyAsnCysGlyAsn                              151015                                                                        ThrGlyHisCysAspThrGlnCysArgAsnTrpGluSerAlaLysHis                              202530                                                                        GlyAlaCysHisLysArgGlyAsnTrpLysCysPheCysTyrPheAsp                              354045                                                                        Cys                                                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      MetIleLeuVal                                                                  (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GluAspAsnGln                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ProAlaGlySerThr                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GlnLysLeuCysGluArgProSerGlyThrTrpSerGlyValCysGly                              151015                                                                        AsnAsnAsnAlaCysLysAsnGlnCysIleAsnLeuGluLysAlaArg                              202530                                                                        HisGlySerCysAsnTyrValPheProAlaHisLysCysIleCysTyr                              354045                                                                        PheProCys                                                                     50                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GluLeuCysGluLysAlaSerLysThrTrpSerGlyAsnCysGlyAsn                              151015                                                                        ThrGlyHisCysAspAsnGlnCysLysSerTrpGluGlyAlaAlaHis                              202530                                                                        GlyAlaCysHisValArgAsnGlyLysHisMetCysPheCysTyrPhe                              354045                                                                        AsnCys                                                                        50                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GluLeuCysGluLysAlaSerLysThrTrpSerGlyAsnCysGlyAsn                              151015                                                                        ThrLysHisCysAspAspGlnCysLysSerTrpGluGlyAlaAlaHis                              202530                                                                        GlyAlaCysHisValArgAsnGlyLysHisMetCysPheCysTyrPhe                              354045                                                                        AsnCys                                                                        50                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GluLeuCysGluLysAlaSerLysThrTrpSerGlyAsnCysGlyAsn                              151015                                                                        ThrLysHisCysAspAsnLysCysLysSerTrpGluGlyAlaAlaHis                              202530                                                                        GlyAlaCysHisValArgSerGlyLysHisMetCysPheCysTyrPhe                              354045                                                                        AsnCys                                                                        50                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      LysThrCysGluAsnLeuSerGlyThrPheLysGlyProCysIlePro                              151015                                                                        AspGlyAsnCysAsnLysHisCysLysAsnAsnGluHisLeuLeuSer                              202530                                                                        GlyArgCysArgAspAspPheXaaCysTrpCysThrArgAsnCys                                 354045                                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AsnLeuCysGluArgAlaSerLeuThrTrpThrGlyAsnCysGlyAsn                              151015                                                                        ThrGlyHisCysAspThrGlnCysArgAsnTrpGluSerAlaLysHis                              202530                                                                        GlyAlaCysHisLysArgGlyAsnTrpLysCysPheCysTyrPheAsp                              354045                                                                        Cys                                                                           (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      AsnThrCysGluAsnLeuAlaGlySerTyrLysGlyValCysPheGly                              151015                                                                        GlyCysAspArgHisCysArgThrGlnGluGlyAlaIleSerGlyArg                              202530                                                                        CysArgAspAspPheArgCysTrpCysThrLysAsnCys                                       354045                                                                        (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      AsnThrCysGluHisLeuAlaAspThrTyrArgGlyValCysPheThr                              151015                                                                        AsnAlaSerCysAspAspHisCysLysAsnLysAlaHisLeuIleSer                              202530                                                                        GlyThrCysHisAspTrpLysCysPheCysThrGlnAsnCys                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      LysThrCysGluLeuAsnAlaAspThrTyrArgGlyProCysPheThr                              151015                                                                        ThrGlySerCysAspAspHisCysLysAsnLysGluHisLeuLeuSer                              202530                                                                        GlyArgCysArgAspAspValArgCysTrpCysThrArgAsnCys                                 354045                                                                        (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      ArgHisCysGluSerLeuSerHisArgPheLysGlyProCysThrArg                              151015                                                                        AspSerAsnCysAlaSerValCysGluThrGluArgPheSerGlyGly                              202530                                                                        AsnCysHisGlyPheArgArgArgCysPheCysThrLysProCys                                 354045                                                                        (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      ArgValCysMetGlyLysSerAlaGlyPheLysGlyLeuCysMetArg                              151015                                                                        AspGlnAsnCysAlaGlnValCysLeuGlnGluGlyTrpGlyGlyGly                              202530                                                                        AsnCysAspGlyValMetArgGlnCysLysCysIleArgGlnCysTrp                              354045                                                                        (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      LysIleCysArgArgArgSerAlaGlyPheLysGlyProCysMetSer                              151015                                                                        AsnLysAsnCysAlaGlnValCysGlnGlnGluGlyTrpGlyGlyGly                              202530                                                                        AsnCysAspGlyProPheArgArgCysLysCysIleArgGlnCys                                 354045                                                                        (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GAGCTTTGCGAGAAGGCTTCTAAGACTTGGTCTGGAAACTGCGGAAACACTGGACATTGC60                GATAACCAATGCAAGTCTTGGGAGGGAGCTGCTCATGGAGCTTGCCATGTTAGAAACGGA120               AAGCATATGTGCTTCTGCTACTTCAACTGC150                                             (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GAGGTTTGCGAGAAGGCTTCTAAGACTTGGTCTGGAAACTGCGGAAACACTGGACATTGC60                (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      GAGCTTTGCGAGAAGGCTTCTAAGACTTGGTCTGGAAACTGCGGAAACACTAAGCATTGC60                GATGATCAATGCAAGTCTTGGGAGGGAGCTGCTCATGGAGCTTGCCATGTTAGAAACGGA120               AAGCATATGTGCTTCTGCTACTTCAACTGC150                                             (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GAGCTTTGCGAGAAGGCTTCTAAGACTTGGTCTGGAAACTGCGGAAACACTAAGCATTGC60                GATAACAAGTGCAAGTCTTGGGAGGGAGCTGCTCATGGAGCTTGCCATGTTAGATCTGGA120               AAGCATATGTGCTTCTGCTACTTCAACTGC150                                             (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 141 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      AAGACTTGCGAGAACCTTTCTGGAACTTTCAAGGGACCATGCATTCCAGATGGAAACTGC60                AACAAGCATTGCAAGAACAACGAGCATCTTCTTTCTGGAAGATGCAGAGATGATTTCNNN120               TGCTGGTGCACTAGAAACTGC141                                                      (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 147 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      AACCTTTGCGAGAGAGCTTCTCTTACTTGGACTGGAAACTGCGGAAACACTGGACATTGC60                GATACTCAATGCAGAAACTGGGAGTCTGCTAAGCATGGAGCTTGCCATAAGAGAGGAAAC120               TGGAAGTGCTTCTGCTACTTCGATTGC147                                                (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      AlaLeuSerCysGlyThrValAsnSerAsnLeuAlaAlaCysIleGly                              151015                                                                        TyrLeuThrGlnAsnAlaProLeuAlaArgGlyCysCysThrGlyVal                              202530                                                                        ThrAsnLeuAsnAsnMetAlaXaaThrThrPro                                             3540                                                                          (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      AlaLeuSerCysGlyThrValAsnSerAsnLeuAlaAlaCysIleGly                              151015                                                                        TyrLeuThrGlnAsnAlaProLeuAlaArgGlyCysCysThrGlyVal                              202530                                                                        ThrAsnLeuAsnAsnMetAlaXaaThrThrPro                                             3540                                                                          (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      GlyIleThrCysGlyMetValSerSerLysLeuAlaProCysIleGly                              151015                                                                        TyrLeuLysGlyGlyProLeuGlyGlyGlySerSerGlyGlyIleLys                              202530                                                                        AlaLeuAsnAlaAlaAlaAlaThrThrPro                                                3540                                                                          (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      ValAspCysGlyGlnValAsnSerSerLeuAlaSerCysIleProPhe                              151015                                                                        LeuThrGlyGlyValAlaSerProSerAlaSerCysCysAlaGlyVal                              202530                                                                        GlnAsnLeuLysThrLeuAlaProThrSerAla                                             3540                                                                          (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      ValLeuThrCysGlyGlnValThrGlyAlaLeuAlaProCysLeuGly                              151015                                                                        TyrLeuArgSerGlnValAsnValProValProLeuThrCysCysAsn                              202530                                                                        ValValArgGlyLeuAsnAsnAlaAlaArgThrThrLeu                                       354045                                                                        (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      AlaLeuAsnCysGlyGlnValAspSerLysAsnLysProCysLeuThr                              151015                                                                        TyrValGlnGlyGlyProGlyGlyProSerGlyLeuCysCysAsnGly                              202530                                                                        ValArgAspLeuHisAsnGlnAlaGlnSerSerGly                                          3540                                                                          (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      AlaIleSerCysGlyGlnValAlaSerAlaIleAlaProCysIleSer                              151015                                                                        TyrAlaArgGlyGlnGlySerGlyProSerAlaGlyCysCysSerGly                              202530                                                                        ValArgSerLeuAsnAsnAlaAlaArgThrThrAla                                          3540                                                                          (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      AAAGAATTCAARYTNTGYSARMGNCC26                                                  (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      AAAGAATTCRTGNGCNGGRAANACRTARTTRC32                                            (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      AAAGAATTCGGNACNTGGWSNGGNGTNTG29                                               (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      AAAGAATTC9                                                                    (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 414 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 16..255                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      GTTTTATTAGTGATCATGGCTAAGTTTGCGTCCATCATCGCACTTCTTTTT51                         MetAlaLysPheAlaSerIleIleAlaLeuLeuPhe                                          1510                                                                          GCTGCTCTTGTTCTTTTTGCTGCTTTCGAAGCACCAACAATGGTGGAA99                            AlaAlaLeuValLeuPheAlaAlaPheGluAlaProThrMetValGlu                              152025                                                                        GCACAGAAGTTGTGCGAAAGGCCAAGTGGGACATGGTCAGGAGTCTGT147                           AlaGlnLysLeuCysGluArgProSerGlyThrTrpSerGlyValCys                              303540                                                                        GGAAACAATAACGCATGCAAGAATCAGTGCATTAACCTTGAGAAAGCA195                           GlyAsnAsnAsnAlaCysLysAsnGlnCysIleAsnLeuGluLysAla                              45505560                                                                      CGACATGGATCTTGCAACTATGTCTTCCCAGCTCACAAGTGTATCTGC243                           ArgHisGlySerCysAsnTyrValPheProAlaHisLysCysIleCys                              657075                                                                        TACTTTCCTTGTTAATTTATCGCAAACTCTTTGGTGAATAGTTTTTATGTAA295                       TyrPheProCys                                                                  80                                                                            TTTACACAAAATAAGTCAGTGTCACTATCCATGAGTGATTTTAAGACATGTACCAGATAT355               GTTATGTTGGTTCGGTTATACAAATAAAGTTTTATTCACCAAAAAAAAAAAAAAAAAAA414                (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      MetAlaLysPheAlaSerIleIleAlaLeuLeuPheAlaAlaLeuVal                              151015                                                                        LeuPheAlaAlaPheGluAlaProThrMetValGluAlaGlnLysLeu                              202530                                                                        CysGluArgProSerGlyThrTrpSerGlyValCysGlyAsnAsnAsn                              354045                                                                        AlaCysLysAsnGlnCysIleAsnLeuGluLysAlaArgHisGlySer                              505560                                                                        CysAsnTyrValPheProAlaHisLysCysIleCysTyrPheProCys                              65707580                                                                      (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 284 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..108                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      GGAAATAATAACGCATGCAAGAATCAGTGCATTCGACTTGAGAAAGCA48                            GlyAsnAsnAsnAlaCysLysAsnGlnCysIleArgLeuGluLysAla                              151015                                                                        CGACATGGGTCTTGCAACTATGTCTTCCCAGCTCACAAGTGTATCTGT96                            ArgHisGlySerCysAsnTyrValPheProAlaHisLysCysIleCys                              202530                                                                        TATTTCCCTTGTTAATTCCATAAACTCTTCGGTGGTTAATAGTGTGCGCATA148                       TyrPheProCys                                                                  35                                                                            TTACATATAATTAATAAGTTTGTGTCACTATTTATTAGTGACTTTATGACATGTGCCAGG208               TATGTTTATGTTGGGTTGGTTGTAATATAAAAAAGTTCACGGATAATAAGATGATAAGCT268               CACGTCGCCAAAAAAA284                                                           (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      GlyAsnAsnAsnAlaCysLysAsnGlnCysIleArgLeuGluLysAla                              151015                                                                        ArgHisGlySerCysAsnTyrValPheProAlaHisLysCysIleCys                              202530                                                                        TyrPheProCys                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      ATAGAATTCGACGTGAGCTTATCATCTTATTATCCG36                                        (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      CTTGGCCTTTGGCACAACTTC21                                                       (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      GCTTTCTCAAGTCTAATGCAC21                                                       (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      AACTCGAGCTGCAGTGTCGACCTATTAACAAGGAAAGTAGC41                                   (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      GGAATAGCCGATCGAGATCTAGGAAACAGCTATGACCATG40                                    (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GGAATAGCCGATCGAGATCTAGGA24                                                    (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 288 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 43..282                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      CCCCGGGCTGCAGGAATTCGCGGCCGCGTTTTATTAGTGATCATGGCTAAGTTT54                      MetAlaLysPhe                                                                  1                                                                             GCGTCCATCATCGCACTTCTTTTTGCTGCTCTTGTTCTTTTTGCTGCT102                           AlaSerIleIleAlaLeuLeuPheAlaAlaLeuValLeuPheAlaAla                              5101520                                                                       TTCGAAGCACCAACAATGGTGGAAGCACAGAAGTTGTGCCAAAGGCCA150                           PheGluAlaProThrMetValGluAlaGlnLysLeuCysGlnArgPro                              253035                                                                        AGTGGGACATGGTCAGGAGTCTGTGGAAACAATAACGCATGCAAGAAT198                           SerGlyThrTrpSerGlyValCysGlyAsnAsnAsnAlaCysLysAsn                              404550                                                                        CAGTGCATTAGACTTGAGAAAGCACGACATGGATCTTGCAACTATGTC246                           GlnCysIleArgLeuGluLysAlaArgHisGlySerCysAsnTyrVal                              556065                                                                        TTCCCAGCTCACAAGTGTATCTGCTACTTTCCTTGTTAATAG288                                 PheProAlaHisLysCysIleCysTyrPheProCys                                          707580                                                                        (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      MetAlaLysPheAlaSerIleIleAlaLeuLeuPheAlaAlaLeuVal                              151015                                                                        LeuPheAlaAlaPheGluAlaProThrMetValGluAlaGlnLysLeu                              202530                                                                        CysGlnArgProSerGlyThrTrpSerGlyValCysGlyAsnAsnAsn                              354045                                                                        AlaCysLysAsnGlnCysIleArgLeuGluLysAlaArgHisGlySer                              505560                                                                        CysAsnTyrValPheProAlaHisLysCysIleCysTyrPheProCys                              65707580                                                                      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We claim:
 1. A recombinant DNA sequence encoding an antimicrobialprotein having an amino acid sequence selected from the group consistingof sequences SEQ ID NO:1 to SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:49,SEQ ID NO:51, SEQ ID NO:59 and SEQ ID NO:37.
 2. A DNA sequence asclaimed in claim 1 which is a cDNA.
 3. A DNA sequence as claimed inclaim 1 which is genomic DNA.
 4. A DNA sequence as claimed in claim 3which is isolated from a plant genome.
 5. A vector containing a DNAsequence as claimed in claim
 1. 6. A cultured cell comprisingrecombinant DNA as claimed in claim 1 such that the encoded protein isexpressed.
 7. A cultured cell as claimed in claim 6 which is amicro-organism.
 8. A cultured cell system as claimed in claim 6 which isa plant cell.
 9. A plant transformed with recombinant DNA as claimed inclaim
 1. 10. Seeds or progeny of a plant as claimed in claim 9, whereinsaid seed or progency comprise said recombinant DNA.
 11. The recombinantDNA sequence of claim 1, wherein said antimicrobial protein is selectedfrom the group consisting of Rs-AFP1, Rs-AFP2, Br-AFP1, Br-AFP2,Bn-AFP1, Bn-AFP2, Sa-AFP1, Sa-AFP2, At-AFP1, Dm-AMP1, Dm-AMP2, Cb-AMP1,Cb-AMP2, Lc-AFP, Ct-AMP1 and Rs-nsLTP.
 12. The recombinant DNA sequenceof claim 1, wherein said antimicrobial protein is isolated from a seedof the family Brassicaceae or of the family Compositae or of the familyLeguminosae.
 13. The recombinant DNA sequence of claim 1, wherein saidantimicrobial protein is isolated from the group consisting of Raphamis,Brassica, Sinapis, Arabidopsis, Dehlia, Cnicus, Lathyrus and Clitoria.